Dissection of Zebrafish Craniofacial Tissues Upon Staining with Alcian Blue

The craniofacial cartilage in vertebrates emerges from a multipotent cell population called the cranial neural crest cells¹. These cells are first specified in the dorsal margins of the neural plate in early embryos, which then migrate large distances to reach the craniofacial region and the pharyngeal arches before differentiating^2,3. The signaling mechanisms that drive this migration and differentiation are largely conserved among vertebrates^4,5. Despite this, the organization, size, and shape of individual cartilage elements are distinct across species, giving rise to diverse craniofacial morphologies⁶. Zebrafish, in particular, has emerged as a powerful model system to study craniofacial morphogenesis, given the transparent nature of the embryos until larval stages. Using simple staining procedures for marking the cartilage, the morphology of many craniofacial structures in zebrafish has been characterized under different perturbations to signaling pathways^7,8,9 as well as when embryos or larvae are exposed to common pollutants present in the environment¹⁰. The Alcian blue staining described here for marking the cartilage involves the use of non-acidic conditions standardized by Walker and Kimmel¹¹, which has been subsequently used by many zebrafish labs working on craniofacial development^{5,12,13,14,15,16,17,18,19}, including the description of usage in high-school settings²⁰. Acid-free staining preserves the bony structures intact, allowing for staining cartilage and bones together using Alcian blue and alizarin red, respectively. However, a detailed protocol for staining as well as for performing precise dissection of the craniofacial cartilages, especially for aiding such studies to be undertaken in resource-limited settings, is missing.

Even though the facial skeletal structure in adult zebrafish is fairly complex, consisting of 43 cartilage-derived bones²¹, the craniofacial cartilage in a larva at 5 days post fertilization (dpf), which has just acquired the ability to feed, is relatively simple and can be broadly divided into a dorsal neurocranium and ventral viscerocranium⁴. These structures support the brain, contribute to the feeding apparatus and give rise to supporting cartilages for gill tissues. The protocol described here will allow for staining zebrafish cartilages with Alcian blue, followed by a detailed procedure for dissecting the neuro and the viscerocranium into separate structures, which will ultimately enable a careful characterization of the shape and size of various cartilages in these structures. As an example, we measure the dimensions of the anterior region of the palate (roof of the mouth) called the ethmoid plate, which is a part of the neurocranium.

The zebrafish maintenance and experimental procedures used in this study were approved by the institutional animal ethics committee, vide Reference TIFR/IAEC/2023-1.

1. Reagent preparation

1. E3 media:
   1. Prepare 50x stock 1 by mixing 3.25 g of sodium phosphate dibasic (0.458 mM Na₂HPO₄), 0.29 g of potassium dihydrogen phosphate (0.042 mM KH₂PO₄), 11.93 g of sodium chloride (4.084 mM NaCl), and 0.48 g of potassium chloride (0.128 mM KCl) in 1 L of distilled water. 
   2. Prepare 50x stock 2 by mixing 2.43 g of calcium chloride (0.33 mM CaCl₂) and 4.07 g of magnesium sulfate (0.33 mM MgSO₄) in 1 L of distilled water.
   3. To prepare 500 mL of 1x E3 media, to 480 mL of distilled water, add 10 mL each of stocks 1 and 2 (pH = 7.4).
      NOTE: Wear gloves during the preparation of these solutions.
2. 4% paraformaldehyde (PFA) in 1x PBS (Phosphate-buffered saline):
   1. Prewarm 100 mL of 1x PBS at 68 °C. Add 4 g of PFA and incubate the solution at 68 °C with periodic mixing until the PFA is completely dissolved.
      NOTE: Wear gloves and a mask while preparing this reagent, preferably under a fume hood as PFA is highly toxic.
3. 0.05% MS-222 (commonly known as tricaine) solution:
   1. Make 4% tricaine stock in E3 and store at 4 °C. To make 10 mL of tricaine stock, add 0.40 g of MS-222 in 7 mL of E3. Shake the tube containing the solution gently to mix and make up the volume to 10 mL. To make 50 mL of 0.05% tricaine solution, add 625 µL of the 4% stock to 49.375 mL of E3.
      NOTE: Wear gloves and a mask while preparing this reagent.
4. 0.4% Alcian blue stock solution in 70% ethanol
   1. To prepare 2 mL of the stock solution, dissolve 8 mg of Alcian blue in 1 mL of 50% ethanol. Heat the solution at 37 °C for 15-20 min with periodic mixing to dissolve. Once the stain dissolves, make up the solution to 70% ethanol by adding 900 µL of 100% ethanol and 100 µL of distilled water.
      NOTE: The stock solution can be stored at room temperature for up to 2 weeks.
5. Stain solution -- 0.02% Alcian blue, 200 mM magnesium chloride (MgCl₂), and 70% ethanol
   1. To prepare 5 mL of Alcian blue stain solution, add 1 mL of 1 M MgCl₂, 3.5 mL of 100% ethanol, and 250 µL of 0.4% Alcian blue stock (8 mg of Alcian blue in 2 mL of 70% ethanol) to 250 µL of water and mix well.
      NOTE: Prepare the stain solution fresh for best staining results.
6. Bleach solution:
   1. Mix equal volumes of 3% hydrogen peroxide (H₂O₂) and 2% potassium hydroxide (KOH). To make 2 mL of bleach solution, mix 1 mL of 3% hydrogen peroxide and 1 mL of 2% potassium hydroxide.
      NOTE: Prepare this solution fresh before beginning this step. Since H₂O₂ is light-sensitive, protect it from being exposed to light as much as possible. Wear gloves while handling H₂O₂ and KOH.
7. Solution I: 20% glycerol + 0.25% KOH
   NOTE: As glycerol is highly viscous, pipette with care. Wear gloves while preparing these solutions.
   1. To prepare 5 mL of solution I, add 2 mL of 50% glycerol and 625 µL of 2% KOH to 2.375 mL of water and mix well.
8. Solution II: 50% glycerol + 0.25% KOH
   1. To prepare 5 mL of solution II, add 3.570 mL of 70% glycerol and 625 µL of 2% KOH to 805 µL of water and mix well.
9. Storage solution: 50% glycerol + 0.1% KOH
   1. To prepare 5 mL of storage solution, add 3.570 mL of 70% glycerol and 250 µL of 2% KOH to 1.180 mL of water and mix well.
10. 3% agarose
    1. Dissolve 3 g of agarose in 100 mL of distilled water. Microwave till the solution becomes clear.
11. 0.1% PBST (0.1% Tween 20 in 1x PBS)
    1. To make 1 L of 1x PBS, add 8 g of NaCl, 0.20 g of KCl, 1.44 g of Na₂HPO₄, and 0.24 g of KH₂PO₄ in 1,000 mL of distilled water. Mix the solution and adjust the pH to 7.4. To make 500 mL of 0.1% PBST, add 5 mL of 10% Tween 20 to 495 mL of 1x PBS.
12. 0.50 µg/mL DAPI solution
    1. Make 10 mg/mL DAPI stock in 1x PBS. To make 10 mL of 0.50 µg/mL DAPI solution, add 0.50 µL of the stock to 10 mL of 1x PBS and shake vigorously to ensure that DAPI dissolves completely.
       NOTE: Wear gloves while preparing these solutions. Protect DAPI from light as it is light-sensitive. The DAPI stock can be stored at 4 °C for more than a year.

2. Embryo collection

1. Set up pairs of male and female zebrafish in breeding tanks the evening before embryos are required, with the male and female fish separated by barriers.
2. Remove the barriers the next morning and collect embryos within 15-20 min after the removal of the barriers.
   NOTE: The timing of barrier removal is helpful in the staging of zebrafish embryos and larvae.
3. Raise the embryos at 28.5 °C until they reach 5 dpf.
   NOTE: Be sure to replace the E3 media with a fresh batch of preheated E3 at 28.5 °C once every 2 days.

3. Alcian blue staining

NOTE: Wear gloves while performing this experiment

1. Anesthetize 20-25 5-day-old zebrafish larvae using 0.05% tricaine in a 2 mL microcentrifuge tube (MCT).
2. After the larvae stop moving, which typically takes ~4-5 min, remove the anesthetic and fix them in 1.5 mL of 4% PFA at room temperature (RT) on a rocker at 60 rpm for 2 h.
3. Remove as much of the fixative as possible using a pipette and add 1.5 mL of 50% ethanol to the sample. Incubate for 10 min while rocking at 60 rpm at RT.
4. Remove as much of the ethanol as possible using a pipette and add 1.5 mL of the Alcian blue stain solution.
5. Incubate the fixed larvae in the stain solution for 18-20 h on a rocker at 60 rpm at RT.
   NOTE: Incubating for a shorter or longer duration of time can lead to certain regions of the cartilage to be understained or overstained, respectively. Here, 18-20 h of staining seemed to work best for clearly visualizing the neuro and the viscerocranium of a 5-day-old larva.
6. Remove the stain solution, add 1.5 mL of distilled water, and incubate for 1-2 min on a rocker at RT.
   NOTE: Staining can be stopped at this step to visualize cell boundaries in the dissected cartilages. However, a disadvantage of stopping the staining protocol at this step without performing subsequent clearing steps is that the boundaries of the neuro and the viscerocranium will still remain slightly blurred, and it becomes more challenging to perform the dissections precisely.
7. Add 1 mL of the bleach solution to the sample and incubate for 20 min at RT in the open without any cover over the plate or wells.
   NOTE: Do not cover the MCTs during this step as the reaction between hydrogen peroxide and potassium hydroxide produces oxygen gas, which could cause the cover to pop off if closed.
8. Remove the bleach solution and perform the following tissue clearing steps.
   1. Add 1.5 mL of solution I to the sample and keep on a rocker at 60 rpm for 40 min at RT.
   2. Remove solution I and add 1.5 mL of solution II to the sample and keep on a rocker at 60 rpm for 2 h at RT.
      NOTE: The clearing steps help remove the excess stain, improving the optical clarity of the samples and thus aiding in performing the dissections efficiently. Handle samples with care while changing solutions, as any damage can affect the future analysis of their morphology.
9. Remove solution II and add 1.5-2 mL of the storage solution. Because of the presence of glycerol, stained samples gradually sink to the bottom within 10 min.
   NOTE: Stained samples can be stored at 4 °C for more than a year.

4. Dissection of the craniofacial skeleton

1. Melt the 3% agarose solution in a microwave until the solution becomes clear.
   NOTE: Do not overheat as the solution can bubble out of the flask. Keep a careful check of the solution while performing this step.
2. Coat a 90 mm Petri dish with 3% agarose by pouring the molten agarose until one third of the depth of the Petri dish is covered and allowing it to solidify. Dissections will be performed in this Petri dish.
   NOTE: The Petri dishes with agarose coating can be prepared a week in advance and stored at 4 °C. The agarose coating provides a softer substrate on which the dissections can be performed without damaging the forceps.
3. Clean the forceps to be used with 70% ethanol and wipe them with a clean piece of standard lab tissue paper.
4. Using a Pasteur pipette, transfer 3-4 stained larvae along with 0.5 mL of storage solution to the agarose-coated petri dish.
   NOTE: The storage solution ensures more optical clarity for performing the dissections compared to doing the same in E3.
5. Orient a stained larva on its lateral side (Figure 1A) and carefully scrape off the yolk from the body of the larva using the forceps
   NOTE: Figure 1B shows an embryo where the yolk has been removed. It is a good practice to transfer the yolk-scraped embryo to a new Petri dish before proceeding with further dissection steps so that the sticky yolk mass floating around does not interfere with the dissection.
6. After the yolk has been removed, hold the larva by its tail region with one forceps and use the other to carefully remove the eyes.
   NOTE: Figure 1C shows an embryo with the yolk and the eyes removed. Eyes should be removed with caution as their proximity to the craniofacial skeleton can interfere with their dissection.
7. Using the forceps, carefully pull and pinch off the brain and other tissues dorsal to the neurocranium. Hold the larva still with one pair of forceps and use the other pair of forceps to make an incision in the middle (Figure 1C, red arrow), followed by pinching off the tissues (Figure 1D).
   NOTE: While performing this step, be careful with removing the tissues closer to the anterior-most regions of the neurocranium, as the cartilage might come off with them.
8. Separate the head of the larva from the rest of the body. The head region after this step primarily contains the neurocranium and the viscerocranium attached to each other at two points-at the anterior and posterior ends-along with a few small unstained tissues attached to these craniofacial structures (Figure 1E).
   NOTE: It is difficult to entirely remove the unstained tissues directly attached to the neurocranium and the viscerocranium. Even though some residual tissues will be present, their presence will not affect any downstream shape or size analysis as they are unstained.
9. Carefully sever the two connections between the neurocranium and the viscerocranium to separate them (Figure 1F, red dashed lines). See Figure 2 for images of the neurocranium and the viscerocranium both in intact as well as dissected embryos.
10. On a clean glass slide, add a drop or two of 100% glycerol. With the help of forceps, carefully transfer the dissected neurocranium or viscerocranium to this slide. Use forceps to gently place a coverslip on these tissues, ensuring no bubbles are formed, and seal the cover slip on all slides with nail polish.
    NOTE: These slides can be stored at 4 °C for more than a year without losing the stain.

5. Quantification of the dimensions of the ethmoid plate

1. Acquire images of the palate using a camera attached to the stereo microscope (Figure 2D). In the absence of a scientific-grade camera, attach a standard smartphone to the stereo microscope to acquire images.
2. For quantification, load the image of a palate into FIJI²² and then, use the line tool to measure the width and the height of the ethmoid plate in microns (see Figure 3A-C) and determine the dimensions using the pixel resolution of the camera.
3. Draw an outline around the ethmoid plate and click on Analyze | Set Measurements | area to obtain the area of the ethmoid plate (Figure 3D,E).

The zebrafish craniofacial skeleton is fully cartilaginous by 5 days post fertilization (dpf) and thus, can be stained using Alcian blue, which is a basic dye capable of binding to glycosaminoglycans present abundantly in cartilages23. This results in the cartilage being stained blue (Figure 1) so that it can be distinguished from other tissues present in the larva using a standard stereo microscope available in most undergraduate lab settings. Once the yolk is scraped off (Figure 1B) and the eyes removed (Figure 1C), the neuro and the viscerocranium can be clearly visualized when viewed from the dorsal and ventral sides of the head, respectively (Figure 2B,C). The neuro and the viscerocranium can then be separated (Figure 2D,E), and individual craniofacial cartilages of interest can then be dissected out, allowing characterization of the shape and dimensions.

Using these simple visualizations of the neuro and the viscerocranium (Figure 2D,E), multiple distinct cartilages can be identified. The anterior neurocranium mainly comprises the palate (Figure 2D) (also called the roof of the mouth), which is analogous to the mammalian secondary palate. The palate in turn can be distinguished into medial and lateral ethmoid plates as well as trabeculae (Figure 2D). Trabeculae are rod-like structures connecting the ethmoid plate to the posterior part of the zebrafish neurocranium, composed of the parachordal cartilages (Figure 2D,E). The zebrafish viscerocranium, on the other hand, consists of a number of cartilage structures, with the anterior-most structure, known as Meckel's cartilage (Figure 2E), which gives rise to the lower jaw. The ethmoid plate and the lower jaw are, in turn, connected through the palatoquadrate and the pterygoid processes, which together enable a coordinated motion of the upper and lower jaw in vertebrates. The viscerocranium, in addition, consists of a number of other cartilages, which support the gill tissues in the fish (Figure 2E).

We next quantified the dimensions for one of the cartilaginous structures, the ethmoid plate, showcasing the usefulness of this staining protocol. For this, we first acquired images of the palate using a camera attached to the stereo microscope (Figure 3A). For quantification, we used FIJI22, a popular freely available image analysis software. The width and the height of the ethmoid plate were 194 ± 1.04 µm and 229 ± 3.13 µm, respectively (errors indicate standard error of the mean; Figure 3C), which can be determined knowing the pixel resolution of the camera. This was extended to obtain the area of the ethmoid plate (25,207 ± 337.61 µm2, Figure 3D,E).

In addition to tissue-scale characterizations, with this simple staining protocol, even cellular arrangements in the palate can be clearly visualized (Figure 3F). When observed closely, consistent with previously published results24, the cellular shapes appear cuboidal in the medial region of the ethmoid plate compared to the lateral regions, where the cells are columnar in shape. Finally, this protocol can be combined with commonly available DAPI stains, which mark the nuclei (Figure 3G). For performing DAPI staining, permeabilize the dissected neurocranium in 0.1% PBST for 45 min, followed by incubation in 0.50 µg/mL DAPI solution for 20 min in the dark. Perform three 15-min-long washes in 0.1% PBST in the dark and mount the sample on a glass slide in 100% glycerol for imaging. A fluorescence microscope is required to image the DAPI-stained samples and following imaging, the number of cells, cellular density, and even shapes of nuclei can be extracted.

Taken together, using a simple staining and dissection protocol that uses minimal resources, both tissue-scale and cellular-scale details can be assessed in the craniofacial region of interest.

Figure 1: Dissection of 5-day-old zebrafish craniofacial skeleton. (A) Zebrafish larva at 5 dpf stained with Alcian blue. (B-E) Steps of dissection. (B) The yolk is first scraped off the embryo. The image shows an embryo with its yolk removed. (C) The eyes are then carefully dissected out. The image shows an embryo with both the yolk and eyes removed. The red arrow indicates the site of incision. (D) Tissues dorsal to the neurocranium, primarily consisting of the brain, are then removed. (E) The craniofacial region is then separated from the rest of the body. (F) Magnified view of the lateral side of the larva with dashed red lines showing where the connections between the neurocranium and the viscerocranium should be severed. In all images, the dashed curves indicate the regions dissected out. Scale bar = 200 µm. Abbreviation: dpf = days post fertilization. Please click here to view a larger version of this figure.

Figure 2: Larval craniofacial skeleton stained with Alcian blue. (A) Dorsal view of the zebrafish head stained with Alcian blue at 5 dpf. (B) Dorsal view of the head with the viscerocranium removed. (C) Ventral view of the head with the neurocranium removed. (D) The dissected neurocranium with the ethmoid plate, trabeculae and parachordal cartilages marked. (E) The dissected viscerocranium with multiple cartilages marked. Scale bar = 100 µm. Abbreviation: dpf = days post fertilization. Please click here to view a larger version of this figure.

Figure 3: Quantification of dimensions of ethmoid cartilage. (A) Neurocranium of a zebrafish larva at 5 dpf. Scale bar = 100 µm. (B) Ethmoid plate with red dashed lines showing the measured length and width. (C) Graph showing the measured length and width of the ethmoid plate in wildtype larvae (n = 15). (D) Ethmoid plate outlined with red dashed lines showing the measured area. (E) Graph showing the measured area. (F) Ethmoid plate with its cell boundaries visible. Red dashed lines mark the boundary of the medial ethmoid plate with the lateral ethmoid plates, where distinct cell shapes can be noted. Scale bar = 50 µm. (G) Ethmoid plate stained with DAPI, a nuclear marker. A confocal microscope with a 20x air objective was used to obtain this sample image, but a wide-field system can be equally used to obtain images of DAPI-stained samples with a slightly lower resolution. Scale bar = 50 µm. Abbreviations: dpf = days post fertilization; p = palate; ep = ethmoid plate; t = trabeculae; Mep = medial ethmoid plate; Lep = the lateral ethmoid plate; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

Craniofacial malformations constitute a majority of all birth defects in humans, often leading to infant mortality. However, the underlying etiology in most craniofacial anomalies remains to be elucidated²⁵. Craniofacial structures are complex in any given vertebrate, and from an undergraduate setting perspective, the first requisite is therefore to have an educational tool that provides students a general overview of distinct structures in the craniofacial region. However, having access to a mammalian embryo in such settings is infeasible in most contexts. Zebrafish, on the other hand, serves as an easier model system to maintain in a small facility²⁶ and has the added advantages of being transparent with a relatively much simpler craniofacial anatomy at early larval stages⁴.

Even though the protocol described in this study is well established and used across most zebrafish labs working on craniofacial morphogenesis, it is yet to be clearly detailed in a simple form, particularly to aid undergraduate labs to take this up as a practical tool. Staining described in this protocol is straightforward and easy to follow, allowing the visualization of craniofacial tissues present deep inside the head, even without them being dissected out. However, this can be used to detect only gross morphological changes; precise characterization of the size and shape of an individual craniofacial structure requires it to be dissected out. The protocol requires only two pairs of forceps and a lab stereoscope using which the entire craniofacial skeleton of zebrafish larvae can be taken out and examined. However, some practice is essential to efficiently perform these dissections. The duration of staining is an important step in the protocol as it allows visualization of components of the craniofacial skeleton with high contrast, further enabling precise dissections. In the case of overstaining, prolonged washes can help remove the excess stain. During the dissection process, careful removal of eyes is vital to make sure that cartilages of interest are still intact.

This method of staining also has a few limitations. One is that Alcian blue dye stains samples slowly, and thus, longer incubation periods are required. Two, because of the dye's tendency to aggregate, stock and stain solutions cannot be stored for long periods. Finally, since the dye is strongly cationic, it can mask anionic sites in antigens, rendering it difficult to combine this protocol with immunohistochemistry.

Even though staining and dissection have been described for 5 dpf larvae in this protocol, the same can be extended to different stages, from 4 dpf when cartilages first form in the zebrafish face, to much later in development. Alcian blue staining can also be combined with alizarin red staining, which stains bones²⁷, thus enabling investigation of skeletal morphology. Importantly, a quantitative analysis can also be performed on the dissected tissues, which was demonstrated through simple measurements performed in the ethmoid plate. This can be extended by employing more sophisticated techniques such as geometric morphometrics, which will allow for a quantitative characterization of tissue shape²⁸. If access to stains such as DAPI is available, then a more nuanced analysis at a cellular scale can be performed in the craniofacial region of interest. Finally, this protocol can be easily adapted to test the impact of various environmental pollutants and teratogens on craniofacial tissues, as previously reported¹⁰. Taken together, this protocol will facilitate hands-on training for undergraduate students, not only for learning to handle embryos from a vertebrate model system but also for careful dissection and analysis of underlying structures in the craniofacial region.